Abstract
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are the molecular correlate of the I h (or I f ) current, which plays a key role in controlling rhythmic activity in cardiac pacemaker cells and spontaneously firing neurons. HCN channels are activated by voltage and modulated by the direct binding of cAMP to their cytoplasmic C-terminal region named CNBD (cyclic nucleotide binding domain). HCN channels are further regulated by TRIP8b, a brain-specific auxiliary subunit which controls the channels’ trafficking and gating. In particular, TRIP8b interacts with the HCN channel CNBD and antagonizes the facilitatory effect of cAMP on channel opening. Recently, we have identified by rational design a 40 aa long peptide (TRIP8b nano ) that recapitulates affinity and gating effects of full length TRIP8b in all HCN isoforms (HCN1, HCN2 and HCN4) and in the cardiac current I f. Guided by a NMR-derived structural model that identifies the key molecular interactions between TRIP8b nano and HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8b nano) which successfully prevented b-adrenergic activation of I f leaving the stimulation of calcium current unaffected. Moreover, we fused TRIP8b nano to an engineered LOV (Light Oxygen Voltage) domain of the plant blue-light receptor phototropin. Our preliminary results show that the synthetic LOV-TRIP8b nano protein is able to inhibit the cAMP regulation of HCN channels in a blue-light dependent manner. TRIP8b nano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.